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CHAPTER 1: An Introduction to Hypothesis-Testing and Experimental Design

Ecologists gain unde standing o! t"e natu al #o ld t" oug" igo ous a$$lication o! t"e scienti!ic %et"od& As 'ou %a' ecall( t"e scienti!ic %et"od inst ucts esea c"e s to )* o+se ,e natu e ca e!ull'( -* as. an inte esting /uestion( and 3* !o %ulate a "'$ot"esis& T"e hypothesis is an e0$licit state%ent as to #"at #e +elie,e to +e t ue a+out t"e o+se ,ed $"eno%ena& T"e scienti!ic %et"od t"en inst ucts esea c"e s to test t"e "'$ot"esis& Testing usuall' in,ol,es designing a %et"od o $ otocol !o collecting in!o %ation 1data* t"at #ill allo# us to e,aluate t"e "'$ot"esis( and !inall'( to acce$t o e2ect it& 3e gene all' !o % a "'$ot"esis +ased u$on o+se ,ations o! natu al $"eno%ena du ing !ield 1o la+o ato '* studies& O!ten t"ese o+se ,ations include t"e detection o! distincti,e $atte ns 1o di!!e ences* in natu e& 4o e0a%$le( 'ou %ig"t notice t"at t"e nu%+e o! $lants di!!e s on no t" and sout"5!acing slo$es& You %ig"t also notice t"at eac" slo$e ecei,es a di!!e ent a%ount o! sunlig"t du ing t"e cou se o! a da'& It is ,e ' i%$o tant t"at t"e o+se ,ed $atte n +e eal and #o t"' o! stud'& T"e e!o e( ecologists a e ca e!ul to %easu e o #uanti$y t"ei o+se ,ations& In ou e0a%$le( 'ou %ig"t count t"e nu%+e o! t ees along t" ee -05% $at"s 1t ansects* on eac" slo$e& You could also /uanti!' t"e nu%+e o! s" u+s in t" ee )5% - a eas 1/uad ates* and %easu e t"e a%ount o! sunlig"t eac"ing t"e g ound using a lig"t %ete & In o de to tell i! t"e t#o slo$es di!!e #it" es$ect to t ee( s" u+s o lig"t( 'ou %ust s"o# t"at t"e di!!e ences 'ou see a e g eate t"an #ould +e e0$ected +' c"ance& T"is is t"e $ o,ince o! statistic analysis and experimental design&


T"e #a' in #"ic" #e dete %ine i! a su+2ecti,e i%$ ession 16educated guess7 o "'$ot"esis* a+out an o+se ,ation is co ect is +' $e !o %ing a "'$ot"esis test& T"is in,ol,es t"e !o %ation o! t#o o %o e "'$ot"eses( collection o! un+iased data( statistical testing( and acce$tance o! one "'$ot"esis&

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T'$icall'( t"e e a e t#o o$$osing "'$ot"eses& T"e null hypothesis 1s'%+olicall' e0$ essed as Ho* is al#a's # itten so t"at it sa's( 6Not"ing out o! t"e o dina ' is going on&7 It "as t"e !ollo#ing !o %8 Ho : Data set A is not di$$erent $rom )is e#ual to* data set +,

9ata set A %a' di!!e slig"tl' ! o% data set B 1!o e0a%$le( t"e %ean o! data set A %a' not +e e0actl' e/ual to t"e %ean o! data set B*( +ut t"is s%all di!!e ence is no g eate t"an t"at e0$ected +' c"ance& T"e alternati-e hypothesis 1s'%+olicall' e0$ essed as HA* is # itten so t"at it sa's( 6So%et"ing odd is going on&7 It "as t"e !ollo#ing !o %8 H A: Data set A is di$$erent $rom )is not e#ual to* data set +,

:nde t"e alte nati,e "'$ot"esis( t"e di!!e ence +et#een data set A and data set B is so g eat t"at it is unli.el' to "a,e occu ed +' c"ance 1!o e0a%$le( t"e %ean o! data set A is t#ice as la ge as t"e %ean o! data set B*& Notice t"at t"e null and alte nati,e "'$ot"eses a e # itten using #o ds t"at indicate %easu e%ents 1i&e&( 6data sets7*& Science al#a's in,ol,es o+2ecti,e %easu e%ents o! t"e eal #o ld& T"e e!o e( "'$ot"eses %ust al#a's +e # itten using #o ds suc" as lengt"( #eig"t( ,olu%e( nu%+e o! eggs( g o#t" ate( etc& To dete %ine #"et"e t"e null o alte nati,e "'$ot"esis is co ect( one %ust conduct a statistical test o! t"e null "'$ot"esis& A statistical test o! t"e null "'$ot"esis esults in eit"e its acce$tance o e2ection& Acce$ting t"e null "'$ot"esis %eans t"at t"e e is no statistical di!!e ence +et#een t"e t#o data sets& Re2ection %eans t"at t"e o+se ,ed di!!e ence is so g eat t"at it is unli.el' to "a,e +een caused +' c"ance& Re2ection o! t"e null "'$ot"esis allo#s one to acce$t t"e alte nati,e "'$ot"esis . It is i%$o tant to eali;e t"at t"e te %s acce$t and e2ect a e used instead o! $ o,e o dis$ o,e in elation to t"e null "'$ot"esis& T"is is +ecause #e a e dealing #it" %at"e%atical $ o+a+ilities( not ce tainties& No matter how great the difference between two data sets, there is still a probability that the difference is due to chance, In ot"e #o ds( i! t"e e is a <=> $ o+a+ilit' t"at t"e e is a di!!e ence( t"en t"e e is also a -> $ o+a+ilit' t"at t"e e is no di!!e ence& So( #e cannot $ o,e o

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dis$ o,e a "'$ot"esis #it" )00> ce taint' +ut #e can %a.e t"e decision( +ased on an o,e #"el%ing $ o+a+ilit'( to acce$t o e2ect a "'$ot"esis&


In +iolog'( e0$e i%ents %a' +e designed to %easu e a $atte n in t"e en,i on%ent o to a$$l' a s$eci!ic t eat%ent to ca e!ull' isolated e0$e i%ental units& E0$e i%ents designed to %easu e $atte ns a e te %ed 6 mensurati-e7? #"e eas e0$e i%ents in,ol,ing t eat%ents a e te %ed 6 manipulati-e7& An experimental unit is t"at 6t"ing7 +eing o+se ,ed o %easu ed . It %a' +e a $lot in t"e !o est( an a/ua iu% !ull o! !is"( o a single "u%an +eing& A treatment is t"e a$$lication o! ce tain conditions 1o !acto s* to t"e e0$e i%ental unit& In %ani$ulati,e e0$e i%ents( t"e in,estigato .ee$s all e0te nal !acto s constant and %ani$ulates onl' t"e desi ed t eat%ent !acto 1s*& In %ensu ati,e e0$e i%ents( t"e in,estigato does not a$$l' t"e t eat%ent& He@s"e %e el' o+se ,es t"e esults o! a t eat%ent $ e,iousl' a$$lied +' Natu e o so%e "u%an distu +ance& In eit"e t'$e o! e0$e i%ent( t"e e!!ects o! t"e t eat%ent on t"e e0$e i%ental unit a e dete %ined +' %easu ing response -aria0les. Conside an e0$e i%ent testing t"e in!luence o! sunlig"t on t"e g o#t" o! suga %a$le 1Acer saccharum* seedlings& An in,estigato esta+lis"es %an' -0- % $lots in t"e !o est and t"ins t"e cano$' t ees to e0$ose so%e $lots to lo# sunlig"t and ot"e s to "ig" sunlig"t& In so%e $lots( t"e cano$' t ees a e not t"inned at all& T"ese $lots a e te %ed 6controls7 +ecause no t eat%ent #as a$$lied& O,e ti%e( t"e in,estigato %easu es t"e "eig"t o! ed $ine seedlings in eac" $lot& Eac" $lot is an e0$e i%ental unit? lig"t intensit' 1o t"inning* is t"e t eat%ent? and c"ange in "eig"t o,e ti%e 1g o#t"* is t"e es$onse ,a ia+le& T"is e0$e i%ent is %ani$ulati,e +ecause t"e in,estigato a$$lied t"inning to s$eci!ic $lots& I! t"e in,estigato "ad %e el' gone out to a !o est and selected $lots t"at "ad ,a 'ing densities o! cano$' t ees 1$e "a$s as a esult o! t ee !all ! o% "ea,' #inds*( t"e e0$e i%ent #ould +e conside ed %ensu ati,e& 3it"in an' $o$ulation( t"e e e0ists a ce tain a%ount o! di!!e ence ! o% one indi,idual o+2ect to t"e ne0t& In t"e $ e,ious e0a%$le( suga %a$le seedlings in a gi,en $lot #ould "a,e a ,a iet' o! "eig"ts 1o g o#t" ates*& T"is -aria0ility can +e t"e esult o! %an' !acto s 1e08 genetics( en,i on%ental in!luences( etc&*& Ou a+ilit'

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to distinguis" +et#een ,a ia+ilit' caused +' a s$eci!ic in!luence 1i&e&( ou t eat%ent* and ,a ia+ilit' caused +' ot"e sou ces is a !unda%ental e/ui e%ent !o a success!ul stud'& T"e sou ces o! ,a ia+ilit' $ esent in an' stud'( ot"e t"an t"e t eat%ent( can %a.e esults di!!icult o i%$ossi+le to inte $ et& T"e $u $ose o! e0$e i%ental design( t"e e!o e( is to %ini%i;e( o "old constant( all ot"e sou ces o! ,a ia+ilit'( except for those that we wish to monitor & T"e !eatu es o! e0$e i%ental design t"at add ess t"e $ o+le% o! ,a ia+ilit' a e sample si1e2 replication2 control2 randomi1ation2 and interspersion, 'A/P%E 'I3E A collection o! %easu e%ents ta.en ! o% an e0$e i%ental unit is te %ed a sample& T"is sa%$le e$ esents a su+set o! t"e $o$ulation +eing studied& In o de to esti%ate t"e ,a ia+ilit' #it"in t"e $o$ulation 1o t eat%ent*( it is necessa ' to "a,e sa%$les ! o% %an' e0$e i%ental units& T"e total nu%+e o! e0$e i%ental units sa%$led is te %ed t"e sample si1e& T"e la ge t"e sa%$le si;e( t"e +ette one is a+le to account !o ,a ia+ilit' #it"in a $o$ulation& In so%e ecological s'ste%s 1e08 la ge !o ests( oceans*( it is i%$ossi+le to get sa%$les ! o% %o e t"an one e0$e i%ental unit& T"e a+ilit' to sa%$le %ulti$le e0$e i%ental units is li%ited +' $ actical const aints 1li%ited $e sonnel( esou ces( ti%e( o %one'*& In t"ese s'ste%s( ,a ia+ilit' %ust +e esti%ated +' sa%$les o,e ti%e and using s$eciali;ed statistics& REP%ICATI! To detect di!!e ences +et#een t eat%ents( it is necessa ' to assess t"e ,a ia+ilit' #it"in eac" t eat%ent& T"e onl' #a' to do t"is is to "a,e %ulti$le e0$e i%ental units !o eac" t eat%ent& T"e sa%$le collected ! o% eac" e0$e i%ental unit #it"in a t eat%ent is te %ed a replicate& 4o t"e $u $oses o! statistical anal'sis( a minimum of three replicates is necessa ' !o eac" e0$e i%ental t eat%ent& In ou $ e,ious e0a%$le on t"e g o#t" es$onse o! suga %a$le seedlings to sunlig"t( t"e e0$e i%ental units to +e e$licated #ould +e t"e $lots o! seedlings e0$osed to eac" le,el o! sunlig"t& It #ould +e ina$$ o$ iate to ta.e se,e al %easu e%ents o! one e0$e i%ental unit 1e&g&( one $lot in lo# sunlig"t* and conside t"e% as e$licates o! t"e enti e t eat%ent le,el& T"is is an e0a%$le o! pseudoreplication.

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C! TR!% T"e cont ol se ,es as a standa d against #"ic" t"e e0$e i%ental units a e co%$a ed& Cont ols a e e0$e i%ental units t"at a e identical to t"ose ecei,ing t eat%ents e0ce$t t"at t"e' do not ecei,e t"e treatment $actor& In ou suga %a$le seedling e0a%$le t"e a%ount o! sunlig"t is t"e t eat%ent( and t"e e!o e #e #ould "a,e one $lot t"at ecei,es no sunlig"t as a cont ol& All ot"e %ani$ulations 1i&e&( #ate ing( !e tili;ation( etc&* s"ould e%ain constant !o all e0$e i%ental units& Cont ols "el$ to identi!' t"ose c"anges t"at %ig"t occu as a esult o! !acto s t"at t"e e0$e i%ente could not "old constant( suc" as dail' te%$e atu e !luctuations& E0$e i%ental units assigned to +e cont ols a e e$licated 2ust as e0$e i%ental units ecei,ing a t eat%ent& I TER'PER'I! A D RA D!/I3ATI! T"e assign%ent o! t eat%ents and cont ols to e0$e i%ental units %ust +e suc" t"at t"e e is no inte action +et#een e0$e i%ental units and t"e e!o e( all units a e li.el'( on a,e age( to ecei,e t"e sa%e en,i on%ental conditions& T"is $lace%ent is e!e ed to as interspersion, T"e #a' in #"ic" inte s$e sion is ac"ie,ed is t" oug" randomi1ation, Ideall'( t"e assign%ent o! t eat%ents and cont ols is done in a $u el' ando% !as"ion #"ic" allo#s c"ance to dete %ine t"ei $lace%ent and to dete %ine #"ic" e0$e i%ental units #ill ecei,e #"ic" t eat%ent& 4o la ge nu%+e s o! e$licates t"is #o .s #ell since it is not $ o+a+le t"at si%ila t eat%ents #ould all end u$ clu%$ed toget"e & 4o s%alle nu%+e s o! e$licates 1as is o!ten t"e case in ecological studies* a systematic alte nating o! t eat%ents and cont ols is o!ten necessa ' 1see 4igu e )&)*& 3"ile desi a+le( inte s$e sion is o!ten not $ossi+le in %ensu ati,e e0$e i%ents&

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R a n d o m ly a s s ig n e d e x p e r im e n ta lu n its

S y s te m a tic a lly a s s ig n e d e x p e r im e n ta lu n its

4igure 1,1

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DI'C"''I! 5"E'TI! ':

)& I! one #e e $lanning to stud' t"e e!!ects o! "ig"#a' salting on st ea% 6"ealt"7( #"at %ig"t 'ou %easu eC 3"at #ould +e 'ou e0$e i%ental unitC -& 3 ite a null and alte nati,e "'$ot"esis !o t"e e!!ect o! "ig"#a' salt on st ea% "ealt"& 3& 3 ite a null and alte nati,e "'$ot"esis !o t"e %a$le seedling e0$e i%ent 1$& 3*& 4& Identi!' t"e $seudo e$lication in t"e situation gi,en +elo#8 In stud'ing t"e e!!ects o! se#age on alga g o#t" in i,e s an in,estigato sets u$ a site a+o,e and +elo# a se#age t eat%ent $lant on a i,e & Se,e al sa%$les a e ta.en u$st ea% o! t"e $lant and se,e al sa%$les a e ta.en do#nst ea%& A& Identi!' sou ces o! ,a ia+ilit' in t"e e0$e i%ent designed to test t"e e!!ects o! sunlig"t on t"e g o#t" o! suga %a$le seedlings 1e0a%$le gi,en in te0t*&

B& You used a ando% %et"od o! inte s$e sing 'ou e0$e i%ental units and ca%e u$ #it" t"e !ollo#ing a ange%ent8

Can 'ou !o esee an' $ o+le% #it" t"is e0$e i%ental designC Ho# %ig"t 'ou sol,e t"is $ o+le%C

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